The present invention relates to a polypeptide of glycosaminoglycan sulfotransferase (glycosaminoglycan sulfate group transferase) and a DNA coding for it. More particularly, the present invention relates to a polypeptide of 6-sulfotransferase originating from human, which sulfates keratan sulfate, but substantially does not sulfate chondroitin, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate and CDSNS-heparin, and a DNA coding for it. The present invention further relates to a method for the preparation of the polypeptide and a method for using the polypeptide of 6-sulfotransferase.
Keratan sulfate is a kind of glycosaminoglycan constituted by galactose residues (Gal), and N-acetylglucosamine residues (GlcNAc) a part of which has substituents of sulfate groups at the C-6 position, and its repetition structure is represented by 3Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw..
It is expected that, if a gene for sulfotransferase for glycosaminoglycan could be cloned, it would provide information about substrate specificity of its receptors and approaches useful for structural and functional investigation of glycosaminoglycan. Various glycosaminoglycan sulfotransferases seem to be involved in the synthesis of glycosaminoglycan. However, it is difficult to clone a cDNA of sulfotransferase.
The present inventors had already purified apparently homogeneously chondroitin 6-sulfotransferase (it may be referred to as "C6ST" hereinafter), which transfers a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the hydroxyl group at C-6 position of N-acetylgalactosamine residue of glycosaminoglycans such as chondroitin, from serum-free chick chondrocyte culture supernatant (Habuchi, O., Matsui, Y., Kotoya, Y., Aoyama, Y., Yasuda, Y., and Noda, M. (1993) J. Biol. Chem. 268, 21968-21974). Moreover, from its partial amino acid sequence, they had prepared an oligonucleotide primer, cloned a chick cDNA, and demonstrated that the polypeptide resulting from the DNA exhibited the C6ST activity. Furthermore, they also found that the enzyme could exhibit activity for transferring sulfate groups to the hydroxyl group at C-6 positions of galactose residues of keratan sulfate (Fukuta, M., Uchimura., K., Nakashima, K., Kato, M., Kimata, K., Shinomura, T., and Habuchi, O. (1995) J. Biol. Chem. 270, 18575-18580).
However, any DNA coding for a polypeptide of sulfotransferase that sulfates only keratan sulfate among chondroitins and keratan sulfate, i.e., sulfotransferase specific for keratan sulfate has not been reported. In particular, such a sulfotransferase originating from human and hence expected to be useful for pharmaceuticals has never been reported.
An enzyme which specifically transfers a sulfate group to keratan sulfate is important in functional studies of keratan sulfate. In particular, such an enzyme of human origin is very important for providing keratan sulfates in order to create pharmaceuticals exhibiting physiological activity preferred for humans. Furthermore, if a DNA coding for a polypeptide of keratan sulfate 6-sulfotransferase originating from human is obtained, it may be expected to be used for therapeutic drugs including those for gene therapy or diagnostic drugs for human diseases caused by, for example, low sulfation of galactose residues of keratan sulfate at their C-6 positions ("low sulfation" herein used means that sulfation degree is low).
The enzyme which specifically transfers a sulfate group to keratan sulfate is expected to be used for the syntheses of GlyCAM-1 (highly glycosylated cell adhesion molecule-1, glycosylation-dependent cell adhesion molecule-1, expected to be used as an antiinflammatory agent), which is considered as one of ligands for L-selectin involved in homing of lymphocytes and rolling of leucocytes occurring during an early phase of inflammation, and sulfated lactosamine oligosaccharides. The DNA coding for this enzyme is also expected to be used for the production of the enzyme in a large scale and in vivo synthesis of GlyCAM-1 by gene transfer.